EFFECTS OF DC FIELDS ON VERTEBRATE NEURONS

直流场对脊椎动物神经元的影响

• 批准号: 6107391
• 负责人: MICHAEL E MCGINNIS
• 金额: $ 5.64万
• 依托单位: SPELMAN COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107391
• 关键词: PC12 cells; biological signal transduction; cell age; cell growth regulation; cell motility; cell type; chick embryo; dendrites; electric field; electrical measurement; electrostimulus; fluorescence microscopy; ganglion cell; image processing; immunocytochemistry; microinjections; nervous system regeneration; neurons; orientation; periaqueductal gray matter; peripheral nervous system disorders; spinal cord injury; statistics /biometry; tissue /cell culture; video microscopy

DC electric fields are thought to stimulate axonal regeneration and growing axons, and so have been suggested as a method for inducing axonal regeneration following nerve injuries. If true, the application of such fields could be important in treating nerve injuries. Although several studies have reported the use of DC fields as a treatment for spinal and peripheral nerve injuries in animals, there are a variety of concerns with each of these studies that have left he general scientific community skeptical of the findings. In contrast, the general phenomena of axonal orientation, growth, and migration towards an electric field in cultaure are well established. While it has been possible to unequivocally demonstrate galvanic responses in vitro, comparable effects have been difficult to produce reliably in vivo. DC fields have low information content and can only provide cells with a directional or stimulatory cue. Either of these cues would be mediated by biasing one side of the cell in terms of ion channels, membrane receptors, membrane potential, etc. In the uniform environment of a culture dish, an applied field may be the only asymmetric influence, and thus may have a significant effect on cellular behavior. In in vivo condiitons, however, there a variety of competing signals for cell behavior such as gradients of diffusible factors, cell to cell interactions, and gradients of substrate adhesiveness. In this situation, the slight directional bias supplied by an electric field may be inconsequential. The long term objectives of the proposed study are to determine the mechanism of the effect of applied fields on cells in vitro and to determine if such a mechanism is functional in the in vivo situation. Specific aims are to; 1. compare the galvanic response of chick neurons grown on simple and complex substrates. 2. determine the effect of developmental stage on the response. 3. examine the effects of cell type and substrate source on the response. 4. to develop the means for systematically and reliable performing such experiments. To test this hypothesis, the galvanic response of neurons will be examined in complex culture conditions such as slice culture. These conditions retain the control, accessibility, and manipulability of the in vitro condition but provide a complex environment ot simulate the natural in vivo situation more closely. The basic method is to acquire time lapse videos of individual cells responding to applied DC fields. The cells will either be fluorescently labeled cells seeded on top of a complex substrate, or cells within a tissue slice that have been dye filled by microinjections. Using fluorescence videomicroscopy, images of individual cells will be made at high magnification. Stepper motors ont he microscope stage will move between many preselected cells and acquire images at regulate time intervals. These images will be recombined into a video sequence detailing the behavior of each cell that will then be statistically analyzed.

直流电场被认为可以刺激轴突再生和生长 轴突等已经被建议作为诱导轴突的方法 神经损伤后的再生。 如果是真的， 电场对治疗神经损伤很重要 尽管几 研究已经报道了使用DC场作为脊柱和脊髓损伤的治疗， 在动物周围神经损伤，有各种各样的关注， 这些研究中的每一项都让科学界 对调查结果持怀疑态度。 相反，轴突的一般现象 在培养中向电场的定向、生长和迁移 都是公认的。 虽然可以明确地 在体外证明了电流反应， 难以在体内可靠地生产。 直流磁场信息量低 内容，只能为细胞提供方向性或刺激性线索。 这两种信号中的任何一种都是通过偏向细胞的一侧来介导的， 离子通道、膜受体、膜电位等术语。 培养皿的均匀环境，应用领域可能是唯一的 不对称的影响，因此可能对细胞的 行为 然而，在体内条件下， 细胞行为的信号，例如扩散因子的梯度、细胞与 细胞相互作用和底物浓度梯度。 在这 在这种情况下，由电场提供的轻微的方向性偏置可以是 无关紧要 拟议研究的长远目标是确定 探讨了电场对体外细胞的作用机制， 确定这种机制在体内情况下是否起作用。 具体目标是： 1. 比较了在简单和 复杂基质 2. 确定发育阶段对反应的影响。 3. 检查细胞类型和底物来源对反应的影响。 4. 开发系统和可靠地执行这些任务的手段， 实验 为了验证这一假设，将检查神经元的电流反应 在复杂的培养条件如切片培养中。 这些条件 保持体外的控制、可及性和可操作性 条件，但提供了一个复杂的环境，以模拟自然的体内 情况更加密切。 基本方法是获取延时视频 单个细胞对直流电场的反应。 这些细胞要么 是接种在复杂基质上的荧光标记细胞，或 通过显微注射填充染料的组织切片内的细胞。 使用荧光视频显微镜，将单个细胞的图像 在高放大率下。 显微镜载物台上的步进电机将移动 并在规定时间内采集图像 的间隔 这些图像将被重新组合成一个视频序列， 然后对每个细胞的行为进行统计分析。